Streptococcus pneumoniae is a common inhabitant of the human upper respiratory tract and is a major pathogen in bacterial pneumonia, otitis media, bacteremia, and meningitis. Between 5 to 70% of all healthy individuals have isolates of Streptococcus pneumoniae. Changes in physical condition and immunological competence as well as the presence of an upper respiratory tract infection often lead to pneumococcal infections (i.e., infections caused by Streptococcus pneumoniae). Pneumococcal disease is ranked among the top ten leading causes of death in the United States. The highest risk groups are children under two years of age, the elderly, and persons with underlying diseases including acquired immunodeficiency syndrome, cirrhosis of the liver, diabetes mellitus, sickle cell disease, splenic dysfunction, acute alcoholism, congestive heart failure, and chronic obstructive pulmonary diseases.
The putative virulence factors of Streptococcus pneumoniae are the capsule, pneumolysin, IgA protease, purpura-producing principle, and neuraminidase. The capsule is a polysaccharide layer which surrounds the bacterium and protects the organism from phagocytosis by macrophages and polymorphonuclear leukocytes and prevents the disposition of complement complexes on the cell wall. Pneumolysin is a pneumococcal protein toxin that inhibits the migration and bactericidal activities of phagocytic cells. It represses lymphokine and the antibody producing ability of lymphocytes, activates the classical complement pathway resulting in depletion of serum opsonic activity and activates the classical complement pathway resulting in depletion of serum opsonic activity. Pneumolysin also causes cell vacuolation of respiratory epithelium structure that leads to cell death. IgA protease may destroy the protection provided by IgA-1 in the mucosa of the respiratory tract. Purpura-producing principle is a cell wall component that causes purpura and dermal hemorrhage in experimental animals. Neuraminidase is a low molecular weight enzyme that cleaves the terminal sialic acid residues from surface glycoproteins and glycolipids of eukaryotic cell membranes. The roles of these virulence factors in the pathogenesis of Streptococcus pneumoniae have not been directly demonstrated.
The most common method of preventing pneumococcal diseases is through antibiotic treatment and vaccination. Penicillin, which has been used for decades in the treatment of pneumococcal disease, has not been shown to improve the survival rate of invasive pneumococcal disease. Due to the wide use of antibiotics and the interspecies transfer of antibiotic resistance gene, penicillin-resistant strains of Streptococcus pneumoniae are common in 25% of the clinical isolates of Streptococcus pneumoniae in the United States. These resistant strains have also caused an outbreak of pneumococcal disease in South Africa. Thus, this wide spread occurrence of resistant Streptococcus pneumoniae strains poses a significant threat to the public health throughout the world.
In addition, continual use of antibiotic treatment of Streptococcus pneumoniae with penicillin may potentiate the development of invasive pneumococcal disease. For example, Streptococcus pneumoniae killed by antibiotics can release cell wall components which can induce meningeal inflammation.
One of the most ideal and effective means of controlling pneumococcal disease is vaccination. The most common vaccine currently used is composed of purified capsular polysaccharides from 23 of the most prevalent strains of Streptococcus pneumoniae; e.g., ACIP (1989) "Pneumococcal polysaccharide vaccine" MMWR 30:64-76; Smart et al. (1987) "New 23-valent pneumococcal vaccine in relation to pneumococcal serotypes in systemic and systemic disease", J. Infect., 14:209-215. Active immunization elicits anticapsular antibodies that opsonize invading pneumococci which are killed by phagocytic cells. This vaccine, however, has several limitations.
First, the elicited anticapsular antibody is type-specific. Currently, there are 84 known different serotypes of Streptococcus pneumoniae and each serotype has a unique capsular structure. Thus, immunization with purified capsular polysaccharides for 23 of the most common strains of Streptococcus pneumoniae only prevents the subsequent infection by those specific pneumococcal serotypes. Therefore, an individual vaccinated by the current vaccine would not be protected from a majority of the known serotypes of Streptococcus pneumoniae that cause a variety of diseases. Field studies have shown that the 23-valent capsular vaccine provides protection against the vaccine-type pneumococcal infection 65% of the time and only protects against all serotypes 0-60% of the time. Second, polysaccharides are not good antigens; especially those isolated from serotypes 6 and 14 of Streptococcus pneumoniae, which are major pathogens in pediatric otitis media and meningitis. Third, the antibody production elicited by polysaccharide is T-cell independent and cannot be enhanced by a second immunization. Forth, the immune response to the current vaccine in the elderly and children under two years of age is weak and cannot protect them against pneumococcal infection.
An alternative approach to protecting the high risk groups from pneumococcal infection would be to isolate pneumococcal proteins that can elicit a protective immune response. Such proteins might serve as a vaccine by themselves or could be used in conjunction with successful polysaccharide vaccines or as carriers of capsular polysaccharides. Pneumolysin and pneumococcal surface protein A (PspA) have both been tested for this purpose. Studies have shown that pneumolysin is present in all serotypes of Streptococcus pneumoniae and exhibits a high degree of conservation in immunological cross-reactivity and nucleotide sequence. Mice immunized with pneumolysin can be rendered inactive by either site-direct or chemical mutagenesis. However, using this attenuated pneumolysin as a pneumococcal vaccine is limited in that it can only be used to halt the progress of disease caused by Streptococcus pneumoniae since pneumolysin is an intercellular protein which is released after an infection with Streptococcus pneumoniae has already occurred.
Other studies have examined pneumococcal surface protein A for its ability to produce protective antibodies. When mice were injected with PspA.sup.+ pneumococci and their isogenic PspA.sup.- mutants, PspA.sup.+ elicited a protective response against fatal doses of encapsulated pneumococci. Using PspA as a vaccine, however, is not effective due to significant differences in PspA-1 among the serotypes of Streptococcus pneumoniae. PspA is polymorphic. There are wide variations among strains in the molecular weight of PspA (60-200 kDa), in the cross-reactivity with monoclonal antibodies, and in the hybridization pattern with a DNA probe that encodes a portion of PspA. When mice were immunized with clones of PspA, a wide range of protection against the challenge of difference serotypes of Streptococcus pneumoniae was found. Thus, the present vaccines do not provide an effective means of preventing pneumococcal diseases.
The present invention solves the problems associated with the present antibiotic and vaccine therapies for pneumococcal diseases (i.e., diseases caused by Streptococcus pneumoniae). The present invention is directed, inter alia, to isolated hemin/hemoglobin-binding proteins of Streptococcus pneumoniae having approximate molecular weights of 18, 43, 55, 66 and 76 kDa, respectively, which are immunogenic and highly conserved among all serotypes of Streptococcus pneumoniae. Unlike the capsular polysaccharides of the 23 strains of Streptococcus pneumoniae described in the prior art, the hemin/hemoglobin-binding proteins described in this invention can serve as antigens for most, if not all, pneumococcal serotypes, are not T-cell independent and are prophylactic against pneumococcal infection. A preferred embodiment of this invention is the isolated hemin/hemoglobin-binding protein of Streptococcus pneumoniae having an approximate molecular weight of 43 kDa. These proteins overcome the limitation associated with Streptococcus pneumoniae antigens known in the art. The present invention provides antibodies to these antigens and compositions containing these antigens. The present invention also provides new vaccines and diagnostic methods including kits to both diagnose and treat human pneumococcal infections.